All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Sucrose is the major transported form of carbohydrates in plants. Carbohydrate exporting tissue is often referred to as source tissue and the importing tissue as sink tissue. The most abundant carbon source transported into legume root nodules is photosynthetically produced sucrose. The transport mechanisms of sucrose in plants have been studied extensively and sucrose transporters from different plant species have been cloned and characterized. For example, the first sucrose transporter, SUT1, was cloned by functional expression in yeast (Riesmeier et al. 1992, EMBO J. 11: 4705-4713). Related genes from plants have since been obtained using the sequence for SUT1, including three genes from tomato (Lycopersicon esculentum). LeSUT1 and its orthologs from other plants are hydrophobic proteins consisting of 12 membrane spanning domains and are located in the plasma membrane of cells mediating highly specific influx of sucrose using a proton-coupled mechanism.
Even though a lot is known about how sucrose is being transported in the plant, less is known about the sucrose distribution in different compartments of the cell. No currently available technology addresses these issues in a satisfactory manner. For example, non-aqueous fractionation is static, invasive, has no cellular resolution and is sensitive to artifacts. While spectroscopic methods such as NMRi (nuclear magnetic resonance imaging) and PET (positron emission tomography) provide dynamic data, they have poor spatial resolution.
The development of genetically encoded molecular sensors, which transduce an interaction of the target molecule with a recognition element into a macroscopic observable signal, via allosteric regulation of one or more signaling elements, may provide answers to some of the questions. The recognition element may simply bind the target, bind and enzymatically convert the target, or may serve as a substrate for the target, as in the use of a specific target sequence in the construction of a protease sensor (Nagai and Miyawaki, 2004). The most common reporter element is a sterically separated donor-acceptor FRET pair of fluorescent proteins (GFP spectral variants or otherwise) (Fehr et al., 2002), although single fluorescent proteins (Doi and Yanagawa, 1999) or enzymes (Guntas and Ostermeier, 2004) are viable, as well. Some molecular sensors additionally employ a conformational actuator (most commonly a peptide which binds to one conformational state of the recognition element), to magnify the allosteric effect upon and resulting output of the reporter element (Miyawaki et al., 1997; Romoser et al., 1997; Kunkel et al., 2004).
The applicability of the method in the absence of a conformational actuator has recently been demonstrated, and its generalizability to a variety of analytes. Members of the bacterial periplasmic binding protein superfamily (PBPs) recognize hundreds of substrates with high affinity (atto- to low micro-molar) and specificity (Tam and Saier, 1993). PBPs have been shown by a variety of experimental techniques to undergo a significant conformational change upon ligand binding; fusion of an individual sugar-binding PBP with a pair of GFP variants produced sensors for maltose, ribose and glucose (Fehr et al., 2002; Fehr et al., 2003; Lager et al., 2003). The sensors were used to measure sugar uptake and homeostasis in living animal cells, and sub-cellular analyte levels were determined with nuclear-targeted versions (Fehr et al., 2004). The successful development of biosensors with bacterial PBPs for maltose, ribose, and glucose suggests to the present inventors that a similar strategy might be adopted to generate a biosensor specific for sucrose if suitable periplasmic sucrose binding proteins (BP) could be identified. A variety of periplasmic sugar binding proteins found in several microorganisms appear to have the potential for the sucrose sensor.
Rhizobium meliloti can occupy at least two distinct ecological niches: in soil as a free-living saprophyte, and as a nitrogen-fixing intracellular symbiont in root nodules of alfalfa and related legumes. AgpA encodes a periplasmic binding protein that is most similar to proteins from the periplasmic oligopeptide binding protein family. It is likely that agpA binds alpha-galactosides because alpha-galactosides induce the expression of agpA, and agpA mutants cannot utilize or transport these sugars. The agpA gene can be down-regulated by the syrA gene products and also by glucose and succinate. Activity of an agpA:TnphoA fusion protein is also downregulated by SyrA. Because syrA is known to be expressed at high levels in intracellular symbiotic R. meliloti and at low levels in the free-living bacteria, it has been hypothesized that agpA may belong to the class of gene products whose expression decreases when R. meliloti becomes an intracellular symbiont (Gage and Long 1998).
The Sinorhizobium meliloti agl operon encodes an alpha-glucosidase and a periplasmic-binding-protein-dependent transport system for alpha-glucosides. (Willis and Walker 1999). A cluster of six genes is involved in trehalose transport and utilization (thu) in Sinorhizobium meliloti. ThuE encodes the binding component of a binding protein-dependent trehalose/maltose/sucrose ABC transporter classified as a trehalose/maltose-binding protein (thuE). When the thuE locus is inactivated by gene replacement, the mutant S. meliloti strain was found to be impaired in its ability to grow on trehalose, and a significant retardation in growth was seen on maltose as well, while the wild type and the thuE mutant were indistinguishable for growth on glucose and sucrose. This suggested a possible overlap in function of the thuEFGK operon with the aglEFGAK operon, which was identified as a binding protein-dependent ATP-binding transport system for sucrose, maltose, and trehalose. ThuE expression is induced only by trehalose and not by cellobiose, glucose, maltopentaose, maltose, mannitol, or sucrose, suggesting that the thuEFGK system is primarily targeted toward trehalose. The aglEFGAK operon, on the other hand, is induced primarily by sucrose and to a lesser extent by trehalose (Jensen et al. 2002).
The Agrobacterium tumefaciens virulence determinant chvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment. The genes were named gguA, -B, and -C, for glucose galactose uptake. Mutations in gguA, gguB, or gguC do not affect virulence of A. tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose (Kemner et al. 1997).
The thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius grows efficiently at 57° C. and pH 3.5. Uptake of radiolabeled maltose was inhibited by maltotetraose, acarbose, and cyclodextrins but not by lactose, sucrose, or trehalose. The corresponding binding protein (AaMalE) interacts with maltose with high affinity (Kd of 1.5 μM). The purified wild-type and recombinant proteins bind maltose with high affinity over a wide pH range (2.5 to 7) and up to 80° C. (Hülsmann et al. 2000).
The extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) from the hyperthermophilic Archaeon Thermococcus litoralis has been crystallized and the structure was determined at 1.85 Å in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter. In vivo, this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced (Diez, 2001).
For none of these proteins had sucrose binding been shown directly. Furthermore, the Agrobacterium homolog of SmThuE had never been analyzed by mutation or by protein analysis. Thus, to develop sensors for sucrose, ThuE was isolated and tested.